1、 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 1,BMS 602A/631: Lecture 4,Light and Fluorescence J.Paul Robinson, PhD Professor of Immunopharmacology and Bioengineering Purdue University www.cyto.purdue.edu,All materials used in this course are available for download on the web a
2、t http:/ 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 2,Absorption,Basic quantum mechanics requires that molecules absorb energy as quanta (photons) based upon a criteria specific for each molecular structure Absorption of a photon raises the molecule from ground state to an ex
3、cited state Total energy is the sum of all components (electronic, vibrational, rotational, translations, spin orientation energies) (vibrational energies are quite small) The structure of the molecule dictates the likely-hood of absorption of energy to raise the energy state to an excited one,3rd E
4、d Shapiro p 84, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 3,Lifetime,Absorption associated with electronic transitions (electrons changing states) occurs in about 1 femtosecond (10-15 s) The lifetime of a molecule depends on how the molecule disposes of the extra energy but
5、many fluorophores have lifetimes around 5 ns Because of the uncertainty principle, the more rapidly the energy is changing, the less precisely we can define the energy So, long-lifetime-excited-states have narrow absorption peaks, and short-lifetime-excited-states have broad absorption peaks,3rd Ed.
6、 Shapiro p 85 4th Ed. Shapiro p 110, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 4,Extinction,Using Beers law (Beer-Lambert law) for light travelling through a curvette thickness d cm containing n molecules/cm3 ln (Io/I) = nd where Io and I are the light entering and leaving a
7、nd is the molecular property called the absorption cross section Now we can state thatln (Io/I) = nd where C is the concentration and a is the absorption coefficient which reflects the capacity of the absorbing substance to absorb light If there are n (molecules/cm3 ; d in cm, must be in cm2 so if i
8、s in cm2/mol, C must be in mol/cm3 do C=a/103 giving log10 (Io/I) = d = A where A is the absorbance or optical densityand is the decadic molar exctinction coeficient in dm3mol-1cm-1,3rd Ed. Shapiro p 86 4th Ed. Shapiro p 111, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 5,Absor
9、bance,O.D. units or absorbance is expressed in logarithmic terms so they are additive. e.g. an object of O.D. of 1.0 absorbs 90% of the light. Another object of O.D. 1.0 placed in the path of the 10% of the light 10% of this light or 1% of the original light is transmitted by the second object It is
10、 possible to express the absorbance of a mixture of substances at a particular wavelength as the sum of the absorbances of the components You can calculate the cross sectional area of a molecule to determine how efficient it will absorb photons. The extinction coefficient indicates this value,3rd ed
11、. Shapiro p 87 4th Ed. Shapiro p 111, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 6,Parameters,Extinction Coefficient refers to a single wavelength (usually the absorption maximum) the cross sectional area of a molecule determines how efficient it will absorb photons Quantum Y
12、ield Qf is a measure of the integrated photon emission over the fluorophore spectral band At sub-saturation excitation rates, fluorescence intensity is proportional to the product of and Qf, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 7,Fluorescence,Quantum Yield,Fluorescence
13、Lifetime () - is the time delay between the absorbance and the emission,kr kr + knr,=,1 kr + knr,=, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 8,Fluorescence,Photon emission as an electron returns from an excited state to ground state, 1990-2005 J.Paul Robinson, Purdue Univer
14、sity Lecture0004.ppt,Slide 9,Fluorescence,Excitation Spectrum Intensity of emission as a function of exciting wavelength (this is the absorbance component) Chromophores are components of molecules which absorb light They are frequently aromatic rings, 1990-2005 J.Paul Robinson, Purdue University Lec
15、ture0004.ppt,Slide 10,Fluorescence,The wavelength of absorption is related to the size of the chromophores Smaller chromophores, higher energy (shorter wavelength), 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 11,Fluorescence,Stokes Shift is the energy difference between the lo
16、west energy peak of absorbance and the highest energy of emission,495 nm,520 nm,Stokes Shift is 25 nm,Fluorescein molecule,Fluorescnece Intensity,Wavelength, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 12,Fluorescence,The longer the wavelength the lower the energy The shorter
17、the wavelength the higher the energy eg. UV light from sun - this causes the sunburn, not the red visible light, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 13, Microsoft Corp, 1995,Electromagnetic Spectrum,Only a very small region within the ES is used for flow cytometry appl
18、ications, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 14,Properties of Fluorescent Molecules,Large extinction coefficient at the region of excitation High quantum yield Optimal excitation wavelength Photostability Excited-state lifetime Minimal perturbation by probe, 1990-2005
19、 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 15,Simplified Jablonski Diagram, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 16,Fluorescence,ENERGY,S0,S1,S2,T2,T1,ABS,FL,I.C.,ABS - Absorbance S 0.1.2 - Singlet Electronic Energy Levels FL - Fluorescence T 1,2 - Corres
20、ponding Triplet States I.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence,IsC,IsC,PH,Vibrational sublevels,Jablonski Diagram,Vibrational energy levels,Rotational energy levels,Electronic energy levels,Singlet States,Triplet States,3rd Ed. Shapiro p 87 4th Ed. Shap
21、iro p 112, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 17,Fluorescence,Stokes Shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission,495 nm,520 nm,Stokes Shift is 25 nm,Fluorescein molecule,Fluorescence Intensity,Waveleng
22、th, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 18,Fluorescence,The longer the wavelength the lower the energy,The shorter the wavelength the higher the energy eg. UV light from sun causes the sunburn not the red visible light, 1990-2005 J.Paul Robinson, Purdue University Lect
23、ure0004.ppt,Slide 19,Fluorescence Excitation Spectra,Intensity related to the probability of the event,Wavelength the energy of the light absorbed or emitted, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 20,Conclusions,Dye molecules must be close to but below saturation levels
24、for optimum emission Fluorescence emission is longer than the exciting wavelength The energy of the light increases with reduction of wavelength, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 21,Relative absorbance of phycobiliproteins,Data from Molecular Probes Website,Phycobil
25、iproteins are stable and highly soluble proteins derived from cyanobacteria and eukaryotic algae with quantum yields up to 0.98 and molar extinction coefficients of up to 2.4 106, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 22,Allophycocyanin (APC),Protein,632.5 nm (HeNe),Exci
26、tation,Emisson,300 nm 400 nm 500 nm 600 nm 700 nm, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 23,Ethidium,PE,cis-Parinaric acid,Texas Red,PE-TR Conj.,PI,FITC,Common Laser Lines, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 24,Quenching, Bleaching & Satur
27、ation,Quenching is when excited molecules relax to ground states via nonradiative pathways avoiding fluorescence emission (vibration, collision, intersystem crossing) Molecular oxygen quenches by increasing the probability of intersystem crossing Polar solvents such as water generally quench fluores
28、cence by orienting around the exited state dipoles,3rd Ed. Shapiro p 90 4th Ed. Shapiro p 115, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 25,Photobleaching,Defined as the irreversible destruction of an excited fluorophore Photobleaching is not a big problem for flow cytometry
29、 as long as the time window for excitation is very short (a few hundred microsconds), 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 26,Photobleaching example from microscopy,FITC - at 4.4 x 1023 photons cm-2 sec-1 FITC bleaches with a quantum efficiency Qb of 3 x 10-5 Therefore
30、FITC would be bleaching with a rate constant of 4.2 x 103 sec-1 so 37% of the molecules would remain after 240 sec of irradiation. In a single plane, 16 scans would cause 6-50% bleaching, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 27,Excitation - Emission Peaks,Fluorophore EX
31、peak EM peak,% Max Excitation at 488 568 647 nm,FITC 496 518 87 0 0 Bodipy 503 511 58 1 1 Tetra-M-Rho 554 576 10 61 0 L-Rhodamine 572 590 5 92 0 Texas Red 592 610 3 45 1 CY5 649 666 1 11 98,Note: You will not be able to see CY5 fluorescenceunder the regular fluorescent microscope becausethe waveleng
32、th is too high., 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 28,Excitation Saturation,The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime f) Optical saturation occurs when the rate of excitation exceeds t
33、he reciprocal of f In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in 1 second requires a dwell time per pixel of 2 x 10-6 sec. Molecules that remain in the excitation beam for extended periods have higher probability of interstate crossings and thus phosphorescence Usually, incre
34、asing dye concentration can be the most effective means of increasing signal when energy is not the limiting factor (i.e. laser based confocal systems), 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 29,Phosphorescence,Following absorption, molecules can relax via a non-radiative
35、 transition to the T1 rather than the S1 state - this is called an intersystem crossing While it is forbidden it does happen and has a low probability and takes a longer time - the energy dissipated is called phosphorescence Phosphorescence has a longer lifetime than fluorescence (milliseconds rathe
36、r than femtoseconds Phosphorescence generally occurs at longer wavelengths than fluorescence because the energy difference between S0 and T1 is lower,3rd Ed Shapiro p 88 4th Ed. Shapiro p 113, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 30,Resonance Energy Transfer,Resonance e
37、nergy transfer can occur when the donor and acceptor molecules are less than 100 of one another (preferable 20-50 ) Energy transfer is non-radiative which means the donor is not emitting a photon which is absorbed by the acceptor Fluorescence RET (FRET) can be used to spectrally shift the fluorescen
38、ce emission of a molecular combination.,3rd Ed. Shapiro p 90 4th Ed. Shapiro p 115, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 31,FRET properties,Isolated donor,Donor distance too great,Donor distance correct, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide
39、 32,Fluorescence,Resonance Energy Transfer, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 33,Raman Scatter,A molecule may undergo a vibrational transition (not an electronic shift) at exactly the same time as scattering occurs This results in a photon emission of a photon differ
40、ing in energy from the energy of the incident photon by the amount of the above energy - this is Raman scattering. The dominant effect in flow cytometry is the stretch of the O-H bonds of water. At 488 nm excitation this would give emission at 575-595 nm,3rd Ed. Shapiro p 93 4th Ed. Shapiro p 118, 1
41、990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 34,Probes for Proteins,FITC 488 525 PE 488 575 APC 630 650 PerCP 488 680 Cascade Blue 360 450 Coumerin-phalloidin 350 450 Texas Red 610 630 Tetramethylrhodamine-amines 550 575 CY3 (indotrimethinecyanines) 540 575 CY5 (indopentamethine
42、cyanines) 640 670,Probe Excitation Emission, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 35,Specific Organelle Probes,BODIPY Golgi 505 511 NBD Golgi 488 525 DPH Lipid 350 420 TMA-DPH Lipid 350 420 Rhodamine 123 Mitochondria 488 525 DiO Lipid 488 500 diI-Cn-(5) Lipid 550 565 di
43、O-Cn-(3) Lipid 488 500,Probe Site Excitation Emission,BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 36,Fluorescence Overlap,488 nm,575 nm,Fluorescein molecule,Flu
44、orescence Intensity,Wavelength (nm),450 500 550 600 650,Overlap of FITC fluorescence in PE PMT,Overlap of PE fluorescence in FITC PMT,PE molecule, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 37,Fluorescence,The longer the wavelength the lower the energy The shorter the wavelen
45、gth the higher the energy eg. UV light from sun - this causes the sunburn, not the red visible light The spectrum is independent of precise excitation line but the intensity of emission is not, 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 38,Mixing fluorochromes,When there are
46、two molecules with different absorption spectra, it is important to consider where a fixed wavelength excitation should be placed. It is possible to increase or decrease the sensitivity of one molecule or another., 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Slide 39,Mixing fluoroch
47、romes,When there are two molecules with different absorption spectra, it is important to consider where a fixed wavelength excitation should be placed. It is possible to increase or decrease the sensitivity of one molecule or another., 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt,Sli
48、de 40,Mixing fluorochromes,When there are two molecules with different absorption spectra, it is important to consider where a fixed wavelength excitation should be placed. It is possible to increase or decrease the sensitivity of one molecule or another., 1990-2005 J.Paul Robinson, Purdue Universit
49、y Lecture0004.ppt,Slide 41,Lecture Summary,Light and Matter Absorption Fluorescence From this lecture you should understand: The nature of fluorescence molecules How fluorescence is generated Why molecules have different excitation and emission What Resonance Energy Transfer is What quantum yield is What fluorescence overlap is How excitation wavelength impacts emission,
